ABSTRACT
A cDNA containing a 813 bp open reading frame encoding vitelline protein BI (FgVPBI) of Fasciola gigantica was cloned. FgVPBI has 96% sequence identity with VPBI of Fasciola hepatica and 84% identity with VPBII F. hepatica. It is far less similar to eggshell precursor proteins of other trematode species, for example, 29% identity with C. sinensis. Northern blot hybridization of total RNA from adult parasites demonstrated a FgVPBI transcript with a size of 1,000 nucleotides. FgVPBI mRNA is localized in the vitelline cells in both vitelline glands and intrauterine eggs. Recombinant FgVPBI was expressed as a 31.5 kDa protein in Escherichia coli and used for production of a polyclonal antiserum in rabbits. The FgVPBI antiserum detected immunoblotted rFgVPBI and native eggshell precursor protein at molecular weights of 31.5 kDa and 31 kDa, respectively. Immunolocalization showed strong staining in the cytoplasm of vitelline cells, in eggshell globules and the shells of eggs.
ABSTRACT
A monoclonal antibody (MoAb) against a recombinant glutathione S-transferase (rGST) of F. gigantica was produced in BALB/c mice. Reactivity and specificity of this monoclonal antibody was assessed by ELISA and immunoblotting. Six stable clones, namely 3A3, 3B2, 3C6, 4A6, 4B1 and 4D6 were obtained, All these MoAb reacted with rGST and native GST at a molecular weight of 28 kDa and found to be IgG1, kappa-light chain isotypes. These MoAb cross-reacted with Schistosoma mansoni and Schistosoma japonicum antigens at molecular weights of 28 and 26 kDa, respectively, but no cross-reactions were detected with antigens of Eurytrema and Paramphistomum spp. The localization of GST in metacercaria, 7-week-old juvenile and adult F. gigantica was performed by immunofluorescence technique, using MoAb as well as polyclonal antibody (PoAb) to the native protein as probes. In general, all clones of MoAb gave similar results and the pattern was quite similar to staining by PoAb. The fluorescence was intense, which implied the presence of a high concentration of GST in the parenchymal tissue in all stages of the parasite. However, the parenchymal cells were not evenly stained which implied the existence of subpopulations of this cell type with regard to GST production and storage. In addition, in adult and juvenile stages a moderate fluorescence was present in the basal layer of the tegument, while light fluorescence was observed in the caecal epithelium, cells in the ovary, testis and vitelline gland of the adult. In the metacercaria stage, in addition to parenchymal tissue, the tegument and tegumental cells were stained relatively more intense with MoAb and PoAb than in other stages.